The respiratory capacity of isolated spinach (Spinacia oleracea L.) chloroplasts, measured as the rate of (14)CO(2) evolved from the oxidative pentose phosphate cycle in darkened chloroplasts exogenously supplied with [(14)C]glucose, was progressively diminished by escalating osmotic dehydration with betaine or sorbitol. Comparing the inhibitions of CO(2) evolution generated by osmotic dehydration in chloroplasts given C-1 and C-6 labeled glucose, 54% and 84% respectively, indicates that osmotic dehydration effects to a greater extent the recycling of the oxidative pentose phosphate intermediates, fructose-6P and glyceraldehyde-3P. Respiratory inhibition in the darkened chloroplast could be alleviated by addition of NH(4)Cl (a stromal alkylating agent), iodoacetamide) an inhibitor of glyceraldehyde-3P dehydrogenase), or glycolate-2P (an inhibitor of phosphofructokinase). It is concluded that the site which primarily mediates respiratory inhibition in the darkened chloroplast occurs at the fructose 1,6-bisphosphatase/phosphofructokinase junction.